Journal of Autism and Childhood Schizophrenia, Vol. 5, No. 2, 1975
Uptake and Efflux of Serotonin from Platelets of Autistic and Nonautistic Children l'~ Arthur Yuwiler, 3 Edward Ritvo, Edward Geller, Ronald Glousman, Gary Schneiderman, and Diane Matsuno Neurobiochemistry Laboratory, Veterans Administration Brentwood Hospital, and the Division of Mental Retardation and Child Psychiatry, University of California, Los Angeles School of Medicine
Th& study examined, in vitro, the uptake and efflux o f serotonin by platelets from autistic children, nonautistic hospitalized comparison cases, and normal children. The autistic patients were carefully selected according to previously established diagnostic criteria. The hospitalized comparison children were utilized to assess possible environmental and dietary influences upon the results. Uptake methods were similar to those used by previous investigators. Two efflux procedures were utilized to explore the possibility that methodological factors accounted f o r previously reported differences between autistic and comparison groups. The results failed to indicate statistically significant differences in uptake or efflux between the autistic and the hospitalized comparison groups or the normals. Methodologic considerations which could possibly account f o r the failure to confirm previous findings are discussed in detail.
Blood serotonin concentrations have been reported to be altered in several childhood diseases (Coleman, 1973). Previous studies from our laboratory (Ritvo, Yuwiler, Geller, Ornitz, Saeger, & Plotkin, 1970) have confirmed 'This research is supported in part by Grants HD 04612, HD 00345, and HD 04364 from the National Institute of Child Health and Human Development. 2The authors are indebted to Gwen McAfee, Selma Plotkin, and Rose Weisler for their assistance. !Requests for reprints should be sent to Dr. Arthur Yuwiler, Neurobiochemistry Laboratory - T-85, Veterans Administration Hospital, Wilshire and Sawtelle Boulevards, Los Angeles, California 90073. 83 9 P l e n u m Publish 9 C o r p o r a t i o n , 2 2 7 West ] 7 t h Street, N e w Y o r k , N . Y . 1 0 0 1 1 . N o part o f this P u b l i c a t i o n m a y be r e p r o d u c e d , stored in a retrieval s y s t e m , or t r a n s m i t t e d , in anY f o r m o r b y a n y means, e l e c t r o n i c , mechanical, p h o t o c o p y i n g , m i c r o f i l m i n g , recording, or o t h e r w i s e , w i t h o u t w r i t t e n permission o f t h e publisher.
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Yuwiler, Ritvo, GeUer, Glousman, Schneiderman, and Matsuno
that children with the syndrome of perceptual inconstancy and diagnosed as having early infantile autism, have higher whole blood serotonin concentrations and platelet counts than age and sex-matched normal comparison groups. The mechanisms responsible for these group differences are unknown and their clinical relevance uncertain. Similarities between the properties of platelets and of monoaminergic storage vesicles (Pletscher, 1968) bave stimulated studies on platelet storage properties in the hope that the platelet might model the serotoninergic neuron. Thus, several investigators have compared monoamine uptake a n d efflux from platelets of autistic and nonautistic children with conflicting results. Lucas, Warner, and Gottlieb (1971) reported no differences in the serotonin uptake of platelets from nonautistic hospitalized children with behavior disorders and brain damage as compared to platelets from those diagnosed as having childhood schizophrenia. Siva Sankar, Cates, Broer, and Sankar (1963) observed decreased serotonin uptake into platelets of children labeled autistic/childhood schizophrenic as compared to nonschizophrenic hospitalized children. Boullin, Coleman, and O'Brien (1970) noted only a slight increase in serotonin uptake, but a markedly elevated efflux, using platelets from six children diagnosed as autistic on the basis of the Rimland E2 Questionnaire (Rimland, 1968) when compared to results with platelets from normals. In a second study Boullin, Coleman, O'Brien, and Rimland (1971) attempted to predict E2 scores exceeding 20 from efflux results, and without knowledge of clinical d a t a . The 5HT efflux of each of 10 patients was paired with that of a normal and a 5HT efflux ratio determined. An efflux ratio in excess of 3:1 was taken to indicate E2 scores exceeding 20. Correct predictions of scores greater than 20 were made in six out of seven cases, and three out of three cases were correctly predicted to be below 20. On this basis they suggested that increased efflux was limited to a specific group of psychotic children with E2 scores over 20, and which were diagnosed on this basis as having primary (Kanner's) infantile autism. The differences in findings between those studies could be the result of variations in patient selection procedures arising from the lack of consensus as to the relevant criteria for diagnosing early infantile autism. This problem continues to plague ail investigators in this area (see discussion section). The differences in findings could also reflect variations in the experimental procedures employed to assess uptake and efflux. For example, Boullin et al. (1971) determined uptake by direct incubation of labeled serotonin with platelet-rich plasma containing EDTA, followed by centrifugation in the cold. Efflux was determined by measuring the loss of labeled serotonin from these platelets after resuspension in homologous platelet-free plasma. Siva Sankar et al. (1963) used heparinized platelets and
Uptake and Efflux of Serotonin
85
determined radioactivity in the washed lysate of cells after incubation. Lucas et al. (1971) used equilibrium dialysis of citrated, platelet-enriched plasma. In the present study we examined whether methodological differences could acount for the conflicting findings in the literature. Two procedures, representing methodological extremes, were employed to assess serotonin uptake and efflux from platelets. The first, based upon removal of unbound serotonin from platelets by dialysis, involved minimal handling of the fragile thrombocytes. Citrate was used as the anticoagulent to minimize changes in platelet morphology and function. The second procedure, derived from that of Boullin et al. (1971), involved centrifugation and resuspension of serotonin loaded platelets with possible attendant clumping, cellular damage, and cellular compression. EDTA was used as anticoagulent. We obviously could not directly assess the possibility that the conflicting results in the literature resulted from differences in diagnostic criteria. However, the experimental population for this study consisted of a carefully evaluated group of children with the syndrome of perceptual inconstancy who clearly met the criteria for autism as originally defined by Kanner. Along with a group of normal children, a group of hospitalized but nonautistic children were also employed as a comparison population to assess the possible influence of hospitalization per se upon the results.
METHODS
Subjects Autistic Patients. The autistic population consisted of 11 boys and 1 girl, all inpatients at the Neuropsychiatric Institute, Division of Mental Retardation and Child Psychiatry, UCLA. Routine evaluations included extensive medical and social histories augmented by a 570-item questionnaire, a minimum of two psychiatric evaluations, psychological testing, two or more EEGs with sleep tracings, skull, chest, and bone-age roentgenograms, routine hemograms, and urine studies. Special blood studies for calcium, uric acid, cholesterol, blood phenylalanine, fasting blood glucose, and aminoacid chromatography were also carried out. Ail autistic patients in this study demonstrated unequivocal evidence of the syndrome of perceptual inconstancy, both on history and when examined immediately prior to testing (Ornitz & Ritvo, 1968; Ornitz, 1973). The criteria necessary for establishing diagnosis on history are: (a) the onset of developmental delays prior to 30 months of age and symptoms indicative
86
Yuwiler, Ritvo, Geller, Glousman, Schneiderman, and Ma|suno
of disturbances of (b) perception and (c) relatedness. All patients also showed disturbances of language (developmental arrests) typically associated with autism. Several had the motility disturbances frequently seen in autistic patients. The signs necessary at the time of this study for inclusion in this group were (a) disturbances o f perception and (b) disturbances of relating. These had to be noted independently by two psychiatrists. The children ranged in age from three to six years with a mean _ standard deviation of 60.2 +_ 31.1 months. Hospitalized Comparison Patients. The hospitalized comparison group consisted of 15 inpatients (7 girls and 8 boys) from the Division of Mental Retardation and Child Psychiatry, UCLA. These patients received the saine clinical and laboratory evaluations as the autistics, had the saine diet, and experienced identical environmental ward conditions. AI1 were diagnosed as having mild to severe mental retardation and developmental delays, and were without evidence of the syndrome of perceptual inconstancy or other signs of autism. Their ages ranged from 5.5 to 11.5 years with a mean +_ standard deviation of 69.4 _ 20.6 months. Normal Comparison Children. The normal comparison children were recruited on a voluntary basis from families, relatives, and friends of the staff. All were developing normally and none had a medical history beyond past minor infectious illnesses. None had histories of diagnosable emotional disturbances or intellectual deficiencies. The ages ranged from 5 to 12 years with a means _+ standard deviation of 97.5 _+ 25.9 months. Ail subjects in [he three groups were free of intercurrent infections and were in good physical health at the time of the study. None had had a febrile illness during the two weeks preceding the study. None of the hospitalized patients had ever received phenothiazine or similar medication.
Procedures Fasting blood was drawn between 7:30 a.m. and 9:00 a.m. from the antecubital vein using plastic syringes. Blood was obtained in one syringe and only rarely was a second venipuncture necessary. Each specimen was distributed into coded tubes containing appropriate anticoagulents: for serotonin (Yuwiler, Plotkin, Geller, & Ritvo, 1970), EDTA, as contained in a B-D Vacutainer tube number 3204 QS; for platelet counts (determined at the Outpatient Clinical Laboratory, UCLA Medical Center, using an Auto Technicon), a second 3240 QS tube; for uptake and efflux studies by Procedure A (see below), 1 ml 3.7% sodium citrate/10 ml blood, by Procedure B, 1 ml 1% NA2 EDTA in .7% NaC1/10 ml blood. The tubes were mixed by very gentle inversion and then kept on ice. Analyses were performed within two hours after the last sample was drawn. To obtain platelet-enriched plas-
Uptake and Efflux of Serotonin
87
ma for uptake and efflux studies the appropriate tubes were centrifuged at 82g for 15 minutes. The supernatant solution was drawn off with a widetipped plastic pipet and transferred to polypropylene test tubes for use in Procedure A or B. ProcedureA (seeFigure 1). Ten microliters of serotonin solution containing. 1/~Ci serotonin-3-1'C (specific activity 5.14 gCi/gmol) in water was added to 1 ml citrated platelet-rich plasma to give a final serotonin concentration of approximately 2 X 10 -5 M. The contents were mixed by gentle inversion, 10/~1 was removed and dissolved in .5 ml Soluene (Packard Instrument Inc., Downers Grove, Illinois). Ten milliliters of a P O P O P - - t o l u e n e scintillation mixture was added and radioactivity determined in a Packard 3375 scintillation counter. The remaining solution was
BLOOD DRAWN IN PLASTIC SYRINGE
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Procedures
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Plotelet counts
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Fig. 1. Outline of procedure.
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88
Yuwiler, Ritvo, Geller, Glousman, Schneiderman, and Matsuno
incubated at 37~ in a water bath under ordinary atmosphere for 90 minutes, except where otherwise stated. At the end of the incubation period, 95 - m l aliquots of the mixture were placed in a dialyzing support as described below, and two .i-ml aliquots were placed in duplicate polypopylene Beckman Microfuge tubes9 Platelets and plasma were separated by centrifugation at approximately 9,000g for two minutes in a Beckman Model 152 Microfuge. Fifty microliters of supernatant plasma were removed, dissolved in Soluene as .described above, and the count rate determined. The remainder of the supernatant was gently removed, the centrifuge tube cut just above the buffy platelet layer, extraneous moisture, when present, was blotted off by inverting the top onto filter paper, and the entire tip containing the platelet pellet was then dropped into .5 ml Soluene. After dissolution of the peUet overnight, the radioactivity was determined as indicated above. Serotonin uptake was calculated both directly from the counts in the pelletted platelets and indirectly from the remaining counts in the supernatant solution. Total counts in the pellet plus supernatant solution were compared to initial counts in the suspension to monitor losses. Dialyzing supports were constructed of 2.5-cm-diameter polyethylene tubing fitted atone end with a stretched single layer of dialyzing tubing held in place with rubber bands. These supports were most conveniently prepared by cutting the closed ends from the plastic outer containers of 20-ml Monojet disposable syringes and carefully smoothing the cut end to prevent injury to the dialyzing tubing. The dialyzing support containing .5 ml incubated platelet-rich plasma (1.2 mm deep) was immersed to a depth of 2.5 mm in a 2-1iter bath of freshly prepared Krebs-Ringer phosphate buffer (Umbreit, Burris, & Stauffer, 1949) containing 7.6 g sodium citrate and omitting calcium. The bath was kept in continuous circulation with magnetic stirrers. After 90 minutes, duplicate. 1-ml aliquots of the platelet-rich plasma within the supports were removed, centrifuged as described above, and count rates determined. Percent efflux was calculated as 100(l-p,/po) where po represents number of micromoles of radioactive serotonin in the platelets before dialysis and p, the number after dialysis. Procedure B (see Figure 1). Ten microliters of serotonin solution containing. 1 laCi serotonin-3-14C (specific activity, 5.14 laCi//amol) in water was added to 1 ml platelet-rich plasma from EDTA-saline-treated blood and the contents were mixed by gentle inversion. A 10-/al aliquot was removed and its radioactivity determined as described above. The remaining solution was incubated at 37~ in a Dubnoff metabolic shaker at 30 excursions/min under 95% O5/5% CO2 at a flow rate of 8 ft3/min. At the end of 90 minutes, except where otherwise stated, the tubes were removed and immediately cooled in ice. Duplicate .1-ml aliquots were taken,
Uptake and Efflux of Serotonin
89
platelets and plasma separated by centrifugation, and radioactivity determined as described above. Uptake was calculated as described in Procedure A. The remaining .8 ml was centrifuged at 6,880g for 5 minutes, the supernatant plasma aspirated, and the walls of the tube wiped free of remaining supernatant solution. Four drops of 1~/0 EDTA/0.7% sodium chloride solution were added and the tube gently agitated to resuspend the platelet pellet by briefly and iightly flicking the base of the tube. The platelets were then resuspended in .8 ml homologous platelet-free plasma (prepared by centrifuging platelet-rich plasma at 20,000g in a Sorvall RC2B refrigerated centrifuge for 10 minutes), inverted, and then briefly mixed with a vortex mixer. Duplicate .1-ml aliquots again were taken for determination of radioactivity in the supernatant plasma and platelets. The remaining solution was replaced in the incubator under O2/CO~ for an additional 90 minutes after which a final duplicate set o f . 1-ml aliquots were removed, centrifuged, and the radioactivity of the supernatant solutions and the platelets determined. Efflux was calculated as 100(l-p,/po) where p, is the radioactivity in the platelets at the end of the efflux period and po is that at the beginning of the efflux period. A second calculation was made from the radioactivity in the supernatant in which efflux was lO0(1-[(S,-So)/po]) where S0 represents the radioactivity in the supernatant at the beginning of the efflux and S, is that at the end of the efflux period. These procedures are summarized in Figure 1.
RESULTS
Uptake Studies Uptake Studies: Procedure A. Figure 2A illustrates typical serotonin uptake by platelet-enriched plasma at 37~ in an air atmosphere. Uptake reached a s~able plateau in 30-60 minutes, remaining relatively unchanged for the next hour, after which platelet radioactivity slowly fell while supernatant radio– rose. Approximately 30~ of the total radioactivity was platelet bound in 90 minutes with a serotonin bath concentration of 2 X 10-5 M. Uptake approximated 1.27/amol/10 ' ' platelets, in agreement with values obtained by others (Lucas et al., 1971; Boullin et al., 1970). This does not represent the maximum uptake capacity of the platelet, however, for an additional 20% is taken into the platelets if the serotonin concentration of the incubation media was inereased twofold (Figure 2A). Conceivably the additional uptake could result from active displacement of endogenous, unlabeled serotonin by radioactive serotonin rather than accumulation, but this seems unlikely since the uptake curve continued to rise smoothly beyond the maximal endogenous content. Figure 3 presents a scatter dia-
90
Yuwiler, Ritvo, Geller, Glousman, Schneiderman, and Matsuno UPTAKE
B
EFFLUX
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Fig. 2. Platelet serotonin uptake and efflux (Procedure A, see text). The curves in A represent the uptake of exogenous "C-labeled serotonin into platelets (open figures and lower curves) and the concomitant loss of serotonin from the plasma (filled circles and upper curve). Uptake curves for the two concentrations of serotonin are shown. The curve for serotonin loss from the plasma is that obtained using a serotonin concentration of 2 X 10-2 M. The curves in B represent the loss of serotonin from platelets (open circles) and supernatant solution (filled circles) in the dialysis tube during the efflux procedure.
gram of the relationship between initial serotonin concentration and ultimate serotonin uptake. The correlation coefficient between these variables is .02, suggesting that uptake is not simply an inverse function of initial serotonin concentration under these conditions. Table I summarizes data obtained from the autistic, normal, and hospitalized nonautistic children using Procedure A. Within the small
Uptake and Efflux of Serotonin
91
populations examined, blood serotonin concentrations were higher in autistic children than in either normals or hospitalized nonautistic subjects. The differences reached statistical significance only between the autistic and normal groups. However, these could be because the samples were so small, because hospitalization may effect blood serotonin concentration, because of the diagnostic nonspecificity of elevated serotonin concentrations, or because of the diagnostic heterogeneity of the populations. Blood platelets were also higher in the autistic group but differences were smaller than previously observed and were not statistically significant. As might be expected from these results, serotonin/10" platelets were also significantly higher in aut9 than in comparison populations but the difference between autistics and hospitalized controls failed to reach statistical significance. I
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Fig. 3, Relationship between basal platelet serotonin content and serotonin uptake under condition of procedure A. Open circles ( o ) are values for normal controls. Open triangles ( A ) are values for hospitalized nonautistic controls, and filled circles (*) are values for autistic subjects. The equation for the least-square line is y = 0 . 0 4 7 x + 1 . 4 0 8 , and the correlation coefficient is .02.
92
Yuwiler, Ritvo, Geiler, Glousman, Schneiderman, and Matsuno Table I, Basal Serotonin, Serotonin Uptake, and Efflux by Platelets
A
Group Normal Autistic Hospital controls
Basal (mean • SE, and N)
Age in months (mean -+SE)
Serotonin (#g/ml)
Platelets (10S/mm s)
5HT/platelet (~mol/1011)
95.5 -+ 25 60.2 • 13 69.4 • 20
.205 -+ .017 (12) .273 • .030(12) a .240 -+ .014(14)
310 -+ 12(14) 333 -+ 23(12) 324 -+21(12)
.369 +- .027(12) .516 +- .060(12) a .436 • .033(12)
B
Group Normal
Procedure A (mean _+SE, and N) Uptake (#mol 5HT/1011 Efflux platelets) (% change)
Procedttre B (mean _+SE, and N) Uptake (btmol 5HT/1011 Efflux platelets) (% change)
1.452 +_.070(11) -22.8 -+ 3.3(11) b 1.482 • .016(11) 2.01 • 2.8(12) b .49 _+ .8(12) c
Autistic
1.221 • .092(11) -22.3 +- 3.2(11) b 1.400 +- .050(11) -.56 • 3,5(12) b .52 • 1.1(12) c Hospitalcontrols 1.309 -+ .020(13) -23.7 -+3.6(13) b Values are given as mean and standard error. The number of patients within each g~oup is indicated within parentheses. Significantly different from normals; p < .05. Calculated from difference in platelet radioactivity between beginning and end of efflux period. CCalculated from dffferences in supernatant radioactivity betwš beginning and end of efflux period.
~
I n a g r e e m e n t with t h e r e p o r t o f Siva S a n k a r (1963), u p t a k e b y p l a t e lets f r o m autistic c h i l d r e n in this h a l f o f t h e s t u d y t e n d e d to be l o w e r t h a n t h o s e f r o m t h e o t h e r g r o u p s . H o w e v e r , t h e d i f f e r e n c e was n o t s t a t i s t i c a l l y significant a n d was n o t o b s e r v e d w h e n r e e x a m i n e d b y P r o c e d u r e B (see below). T h e d i f f e r e n c e was also d i m i n i s h e d w h e n u p t a k e was c a l c u l a t e d f r o m t h e loss o f r a d i o a c t i v i t y o f t h e s u p e r n a t a n t d u r i n g i n c u b a t i o n r a t h e r t h a n f r o m t h e g a i n in p l a t e l e t r a d i o a c t i v i t y . B a s œ o n c h a n g e s in t h e s u p e r n a t a n t , p l a t e l e t u p t a k e was 1 . 2 6 5 - . 1 7 7 / a m o l / 1 0 " platelets ( m e a n -+ SE) for autistics, 1.346 +-- .155 for h o s p i t a l i z e d c o n t r o l s , a n d 1.322 +- .160 f o r n o r m a l s . Because o f the ease o f s a m p l i n g , s u p e r n a t a n t c o u n t s are o f t e n u s e d in u p t a k e studies. This has the d i s a d v a n t a g e t h a t c a l c u l a t i o n s a r e b a s e d u p o n the d i f f e r e n c e b e t w e e n t w o large n u m b e r s a n d is s u b j e c t t o t h e e r r o r s in b o t h . D i r e c t e s t i m a t i o n s o f r a d i o a c t i v i t y in platelets a v o i d this s o u r c e o f e r r o r b u t surfer f r o m p o s s i b l e s a m p l i n g e r r o r s d u e t o t h e p e n c h a n t o f p l a t e lets to a d h e r e to surfaces a n d f r o m v a r i a b l e e n t r a p m e n t o f s u p e r n a t a n t in the p l a t e l e t pellet.
Uptake and Efflux of Serotonin
93
Uptake Studies: Procedure B. A typical uptake curve under the conditions described in Procedure B is presented in Figure 5A while Figure 5B presents the temporal changes in platelet content following resuspension o f loaded platelets in h o m o l o g o u s platelet-free plasma. Serotonin uptake continued to rise over the 90-minute incubation period and had not reached plateau values by the end of that time. Nonetheless, 90 minutes was selected as the end point o f uptake time to c o n f o r m as closely as possible to the procedure described by Boullin et al. (1970). The results obtained are summarized in Table IB. Serotonin uptake by platelets in Procedure B was slightly higher than that observed using Procedure A. The groups did not differ on this measure and uptakes were similar to those reported by Boullin et al. (1970). As seen in Figure 6, there was I
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Fig. 4. Relationship between serotonin uptake by platelets and serotonin loss from platelets during dialysis (Procedure A). Open circles represent values for normal control children, open triangles are those for nonautistic hospitalized controls, and fiUed circles are those for autistic subjects. The equation for the least-squares line is y = 12.5 x + 5.3, and the correlation coefficient is .39.
94
Yuwiler, Ritvo, Geller, Glousman, Schneiderman, and Matsuno 20 UPTAKE
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Fig. 5. Platelet serotonin uptake and efflux. A represents the uptake of exogenous ;'C-labeled serotonin into platelets (open circles) and the concommitant loss from platelets (filled circles)under the incubation conditions described in Procedure B. B represents the leakage from platelets (open circles) and the increase in plasma upon resuspension and incubation of serotonin-loaded platelets with homologous platelet-free pJasma (filled circles) as described in Procedure B. a g a i n little r e l a t i o n s h i p b e t w e e n s e r o t o n i n u p t a k e a n d b a s a l platelet serotonin content.
Efflux Studies Efflux Studies: Procedure A. A typical curve for the efflux o f seroton i n f r o m p r e l o a d e d platelets as m e a s u r e d using the dialysis t e c h n i q u e is s h o w n i n Figure 2B. F i n a l platelet s e r o t o n i n c o n c e n t r a t i o n is 60--100070 o f
Uptake and Efflux of Serotonin
95
the initial loaded level. Presumably, the initial loss is diffusible serotonin from the cytoplasm while the stabilized level consists of vesicular-bound serotonin.. Again, however, loss is n o t a simple function of initial serotonin content. Thus the scatter diagram relating uptake to efflux (Figure 4) indicates that only 16070 of the variance, at most, can be accounted for by the platelet serotonin content at the end of the uptake period. As seen in Table 1B, groups did hOt differ in serotonin uptake using this procedure. Efflux Studies: Procedure B. Efflux of serotonin from platelets in Procedure B was minimal whether calculated from the loss of platelet serotonin before and after reincubation in homologous plasma or from the increase in supernatant radioactivity. However, a mean loss of radioactivity of 26.907o for platelets of autistic children and 27.7070 for platelets of normals was observed during the concentration and resuspension of preloaded platelets. This loss in platelet radioactivity is equivalent to that seen in ProI
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BASAL SEROTONIN CONTENT (#mol serotonin/lO 11 platelets) Fig. 6. Relationship between basal serotonin platelet content and serotonin uptake under the incubation conditions of Procedure B. Open circles represent values for normal control children; filled circles, values for autistic children. The equation of the least-square line is y = 1.534 x + .912, and the correlation coefficient is .34.
96
Yuwiler, Ritvo, Geller, Glousman, Schneiderman, and Matsuno
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Fig. 7. Relationship betwee¡ serotonin uptake and serotonin efflux from platelets under the condition described for Procedure B. Open circles represent values for normal controls, while filled circles are those for autistic subjects. The equation for the least-squares fit of the data is y = -1.83 x + 3.38, and the correlation coefficient is -.27.
cedure A during dialysis. As seen in Table IB, efflux was the same for both groups by this procedure. Again, the scatter diagram relating efflux and uptake, Figure 7, suggests that these two processes are only minimally related.
DISCUSSION
Several variables could contribute to differences others have observed in serotonin uptake and efflux from platelets of autistic and normal children. Among these are diagnostic criteria, age, prior drug treatment, and experimental methodology. The diagnostic comparability of the experimental groups is particularly difficult to assess. Siva Sankar et al. (1963) labeled their experimental population autistic/childhood schizophrenic but did not specify their diagnostic criteria. Lucas et al. (1971) used the criteria
Uptake and Efflux of Serotonin
97
of Bender (1968) and Rabinovich, Lucas, Ingram, and Shaw (1965) to define their experimental population as schizophrenics while Boullin et al. (1970, 1971) used the experimental history checklist of Rimland (1968) to identify patients as havingprimary infantile autism. The criteria used in the present study have been detailed in the liter” (Ornitz & Ritvo, 1968) and are summarized in the Methods section. The prior drug status of the patients also varies between studies. Experimental and comparison subjects in Siva Sankar's study (1963) were drug free for 7-10 days prior to the experiment. Those in Lucas' study (1971) were drug free for "at least three days.', Boullin's (1970) patients were drug free for ai least one month. In the present study, none of the subjects had ever received major tranquilizers and ail of them were free of ail medication for at least three weeks prior to testing. Age is also an important experimental variable since both blood serotonin content and platelet counts decrease with age until adult levels are reached at age 14 (Ritvo et al., 1970). Siva Sankar's patients (1963) were 7-15 years of age and differences in uptake were observed only in the 10-15-year-old group. Lucas' patients (1971) ranged from 8 to 15 years of age, with a mean of 12.9. Boullin's patients (1970) ranged in age from 4 to 14 years. In the present study the autistics' mean age was 60.2 months (range 3-6 years), the normals' was 97.5 months (range 5-12 years), and the hospitalized controls 69.4 months (range 5.5-11.5 years). The effects of anticoagulents and temperature on platelet function and morphology are known to be complex. Siva Sankar et al. (1963) used heparin as anticoagulent, Lucas et al. (1971) used sodium citrate a t a final concentration of 1.8o70, and Boullin et al. (1970) used 1~ EDTA. In the present study, citrate at a final concentration of .38~ was used for Procedure A, and 1.8~ EDTA was used in Procedure B. Platelets in citrate are generally considered to maintain normal morphology. Although citrate inhibits dumping, clumping can occur with low levels of citrate (or heparin). Further, platelet aggregation in citrate solutions is induced by sudden chilling, particularly if followed by stirring during rewarming (Kattlove & Alexander, 1971). Indeed, resuspension of packed, chilled, citrated platelets leads to massive loss of label. Chilling itself leads to platelet sphering and an increase in platelet volume, while r and washing often result in platelet damage and release of ADP. Platelets treated with EDTA do hOt clump upon centrifugation and chilling, although they may be otherwise damaged by the procedure. However, EDTA alone causes platelet rounding, swelling, and an increase in platelet volume. In the studies discussed here, Siva Sankar et al. centrifuged and washed heparinized serotoninloaded platelets before lysis and counting. Lucas et al. cooled and centrifuged citrated platelets and maintained them a t . 1~ before rewarming and dialyzing. Boullin used EDTA, chilling, and centrifugation, before resus-
98
Yuwiler, Ritvo, Geller, Glousman, Schneiderman, and Matsuno
pension and reincubation at 37~ These differences in technique might yield platelet preparations with different metabolic transport properties. In summary, platelets from young normals, autistics, or hospitalized controls did not differ in serotonin uptake or efflux in two procedures which differed in anticoagulent, gas phases, temperature, and platelet treatment. As detailed elsewhere (Ornitz & Ritvo, 1968; Ornitz, 1973) confusion exists in the literature over the nature and diagnosis of autism and while we believe that a neuropathophysiologic substrate is involved in this disease, the present results do not support the view that this is reflected in the monamine transport system of platelets.
REFERENCES Bender, L. Childhood schizophrenia: A review. International Journal of Psyehiatry, 1968, 5, 211. Boullin, D. J., Coleman, M., & O'Brien, R. A. Abnormalities in platelet 5-hydroxytryptamine efflux in patients with infantile autism. Nature, 1970, 226, 371-372. Boullin, D. J., Coleman, M., O'Brien, R. A., & Rimland, B. Laboratory predictions of infantile autism based on 5-hydroxytryptaminœ efflux from blood platelets and their correlation with the Rimland E2 score. Journal of Autism and Childhood Schizophrenia, 1971, 1, 63-71. Coleman, M. (Ed.). Serotonin in Down's Syndrome. New York: American Elsevier, 1973. Chap. 9. Kanner, L. Autistic disturbances of affective contact. Nervous Child, 1943, 2, 217-250. Kattlove, H. E., & Alexander, B. The effect of cold on platelets: Cold-induced platelet aggregation. Blood, 1971, 38, 39-48. Lucas, A. R., Warner, K., & Gottlieb, J. S. Biological studies in childhood schizophrenia. Biological Psychiatry, 1971, 3, 123-128. Ornitz, E. M. Childhood autism: A review of the clinical and experimental literature (medical progress). California Medicine, 1973, 118, 21-47. Ornitz, E. M., & Ritvo, E. R. Perceptual inconstancy in early infantile autism. Archives of General Psychiatry, 1968, 18, 76-98. Pletscher, A. Metabolism, transfer and storage of 5-hydroxytryptamine in blood platelets. British Journal of Pharmaeology and Chemotherapy, 1968, 32, 1-16. Rabinovitch, R. D., Lucas, A. R., Ingram, W., & Shaw, C. R. Childhood schizophrenia: Evolution to adulthoocl. Presented at American Orthopsychiatric Association Annual Mš New York, 1965. Rimland, B. On the objective diagnosis of infantile autism. Acta Paedopsyehiatriea, 1968, 35, 1946-1961. Ritvo, E. R., Yuwiler, A., Geller, E., Ornitz, E. M., Saeger, K., & Plotkin, S. Increased blood serotonin and platelets in early infantile autism. Archives ofGeneral Psyehiatry, 1970, 23, 566-572. Siva Sankar, D. V., Cates, N., Broer, H., & Sankar, D. B. Biochemical parameters of childhood schizophrenia (autism) and growth. In J. Wortis (Ed.), Recent Advances in Biologieal Psychiatry (Vol. 5). New York: Plenum, 1963, pp. 76-83. Umbreit, W. W., Burris, R. H., & Stauffer, T. D. Mannometric techniques. Minneapolis: Burgess, 1949. Yuwiler, A., Plotkin, S., Geller, E., & Ritvo, E. Rapid accurate procedure for the determination of serotonin in whole human blood. Biochemical Medicine, 1970, 3, 426-436.
Uptake and Efflux of Serotonin from Platelets of Autistic and Nonautistic Children l'~ Arthur Yuwiler, 3 Edward Ritvo, Edward Geller, Ronald Glousman, Gary Schneiderman, and Diane Matsuno Neurobiochemistry Laboratory, Veterans Administration Brentwood Hospital, and the Division of Mental Retardation and Child Psychiatry, University of California, Los Angeles School of Medicine
Th& study examined, in vitro, the uptake and efflux o f serotonin by platelets from autistic children, nonautistic hospitalized comparison cases, and normal children. The autistic patients were carefully selected according to previously established diagnostic criteria. The hospitalized comparison children were utilized to assess possible environmental and dietary influences upon the results. Uptake methods were similar to those used by previous investigators. Two efflux procedures were utilized to explore the possibility that methodological factors accounted f o r previously reported differences between autistic and comparison groups. The results failed to indicate statistically significant differences in uptake or efflux between the autistic and the hospitalized comparison groups or the normals. Methodologic considerations which could possibly account f o r the failure to confirm previous findings are discussed in detail.
Blood serotonin concentrations have been reported to be altered in several childhood diseases (Coleman, 1973). Previous studies from our laboratory (Ritvo, Yuwiler, Geller, Ornitz, Saeger, & Plotkin, 1970) have confirmed 'This research is supported in part by Grants HD 04612, HD 00345, and HD 04364 from the National Institute of Child Health and Human Development. 2The authors are indebted to Gwen McAfee, Selma Plotkin, and Rose Weisler for their assistance. !Requests for reprints should be sent to Dr. Arthur Yuwiler, Neurobiochemistry Laboratory - T-85, Veterans Administration Hospital, Wilshire and Sawtelle Boulevards, Los Angeles, California 90073. 83 9 P l e n u m Publish 9 C o r p o r a t i o n , 2 2 7 West ] 7 t h Street, N e w Y o r k , N . Y . 1 0 0 1 1 . N o part o f this P u b l i c a t i o n m a y be r e p r o d u c e d , stored in a retrieval s y s t e m , or t r a n s m i t t e d , in anY f o r m o r b y a n y means, e l e c t r o n i c , mechanical, p h o t o c o p y i n g , m i c r o f i l m i n g , recording, or o t h e r w i s e , w i t h o u t w r i t t e n permission o f t h e publisher.
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Yuwiler, Ritvo, GeUer, Glousman, Schneiderman, and Matsuno
that children with the syndrome of perceptual inconstancy and diagnosed as having early infantile autism, have higher whole blood serotonin concentrations and platelet counts than age and sex-matched normal comparison groups. The mechanisms responsible for these group differences are unknown and their clinical relevance uncertain. Similarities between the properties of platelets and of monoaminergic storage vesicles (Pletscher, 1968) bave stimulated studies on platelet storage properties in the hope that the platelet might model the serotoninergic neuron. Thus, several investigators have compared monoamine uptake a n d efflux from platelets of autistic and nonautistic children with conflicting results. Lucas, Warner, and Gottlieb (1971) reported no differences in the serotonin uptake of platelets from nonautistic hospitalized children with behavior disorders and brain damage as compared to platelets from those diagnosed as having childhood schizophrenia. Siva Sankar, Cates, Broer, and Sankar (1963) observed decreased serotonin uptake into platelets of children labeled autistic/childhood schizophrenic as compared to nonschizophrenic hospitalized children. Boullin, Coleman, and O'Brien (1970) noted only a slight increase in serotonin uptake, but a markedly elevated efflux, using platelets from six children diagnosed as autistic on the basis of the Rimland E2 Questionnaire (Rimland, 1968) when compared to results with platelets from normals. In a second study Boullin, Coleman, O'Brien, and Rimland (1971) attempted to predict E2 scores exceeding 20 from efflux results, and without knowledge of clinical d a t a . The 5HT efflux of each of 10 patients was paired with that of a normal and a 5HT efflux ratio determined. An efflux ratio in excess of 3:1 was taken to indicate E2 scores exceeding 20. Correct predictions of scores greater than 20 were made in six out of seven cases, and three out of three cases were correctly predicted to be below 20. On this basis they suggested that increased efflux was limited to a specific group of psychotic children with E2 scores over 20, and which were diagnosed on this basis as having primary (Kanner's) infantile autism. The differences in findings between those studies could be the result of variations in patient selection procedures arising from the lack of consensus as to the relevant criteria for diagnosing early infantile autism. This problem continues to plague ail investigators in this area (see discussion section). The differences in findings could also reflect variations in the experimental procedures employed to assess uptake and efflux. For example, Boullin et al. (1971) determined uptake by direct incubation of labeled serotonin with platelet-rich plasma containing EDTA, followed by centrifugation in the cold. Efflux was determined by measuring the loss of labeled serotonin from these platelets after resuspension in homologous platelet-free plasma. Siva Sankar et al. (1963) used heparinized platelets and
Uptake and Efflux of Serotonin
85
determined radioactivity in the washed lysate of cells after incubation. Lucas et al. (1971) used equilibrium dialysis of citrated, platelet-enriched plasma. In the present study we examined whether methodological differences could acount for the conflicting findings in the literature. Two procedures, representing methodological extremes, were employed to assess serotonin uptake and efflux from platelets. The first, based upon removal of unbound serotonin from platelets by dialysis, involved minimal handling of the fragile thrombocytes. Citrate was used as the anticoagulent to minimize changes in platelet morphology and function. The second procedure, derived from that of Boullin et al. (1971), involved centrifugation and resuspension of serotonin loaded platelets with possible attendant clumping, cellular damage, and cellular compression. EDTA was used as anticoagulent. We obviously could not directly assess the possibility that the conflicting results in the literature resulted from differences in diagnostic criteria. However, the experimental population for this study consisted of a carefully evaluated group of children with the syndrome of perceptual inconstancy who clearly met the criteria for autism as originally defined by Kanner. Along with a group of normal children, a group of hospitalized but nonautistic children were also employed as a comparison population to assess the possible influence of hospitalization per se upon the results.
METHODS
Subjects Autistic Patients. The autistic population consisted of 11 boys and 1 girl, all inpatients at the Neuropsychiatric Institute, Division of Mental Retardation and Child Psychiatry, UCLA. Routine evaluations included extensive medical and social histories augmented by a 570-item questionnaire, a minimum of two psychiatric evaluations, psychological testing, two or more EEGs with sleep tracings, skull, chest, and bone-age roentgenograms, routine hemograms, and urine studies. Special blood studies for calcium, uric acid, cholesterol, blood phenylalanine, fasting blood glucose, and aminoacid chromatography were also carried out. Ail autistic patients in this study demonstrated unequivocal evidence of the syndrome of perceptual inconstancy, both on history and when examined immediately prior to testing (Ornitz & Ritvo, 1968; Ornitz, 1973). The criteria necessary for establishing diagnosis on history are: (a) the onset of developmental delays prior to 30 months of age and symptoms indicative
86
Yuwiler, Ritvo, Geller, Glousman, Schneiderman, and Ma|suno
of disturbances of (b) perception and (c) relatedness. All patients also showed disturbances of language (developmental arrests) typically associated with autism. Several had the motility disturbances frequently seen in autistic patients. The signs necessary at the time of this study for inclusion in this group were (a) disturbances o f perception and (b) disturbances of relating. These had to be noted independently by two psychiatrists. The children ranged in age from three to six years with a mean _ standard deviation of 60.2 +_ 31.1 months. Hospitalized Comparison Patients. The hospitalized comparison group consisted of 15 inpatients (7 girls and 8 boys) from the Division of Mental Retardation and Child Psychiatry, UCLA. These patients received the saine clinical and laboratory evaluations as the autistics, had the saine diet, and experienced identical environmental ward conditions. AI1 were diagnosed as having mild to severe mental retardation and developmental delays, and were without evidence of the syndrome of perceptual inconstancy or other signs of autism. Their ages ranged from 5.5 to 11.5 years with a mean +_ standard deviation of 69.4 _ 20.6 months. Normal Comparison Children. The normal comparison children were recruited on a voluntary basis from families, relatives, and friends of the staff. All were developing normally and none had a medical history beyond past minor infectious illnesses. None had histories of diagnosable emotional disturbances or intellectual deficiencies. The ages ranged from 5 to 12 years with a means _+ standard deviation of 97.5 _+ 25.9 months. Ail subjects in [he three groups were free of intercurrent infections and were in good physical health at the time of the study. None had had a febrile illness during the two weeks preceding the study. None of the hospitalized patients had ever received phenothiazine or similar medication.
Procedures Fasting blood was drawn between 7:30 a.m. and 9:00 a.m. from the antecubital vein using plastic syringes. Blood was obtained in one syringe and only rarely was a second venipuncture necessary. Each specimen was distributed into coded tubes containing appropriate anticoagulents: for serotonin (Yuwiler, Plotkin, Geller, & Ritvo, 1970), EDTA, as contained in a B-D Vacutainer tube number 3204 QS; for platelet counts (determined at the Outpatient Clinical Laboratory, UCLA Medical Center, using an Auto Technicon), a second 3240 QS tube; for uptake and efflux studies by Procedure A (see below), 1 ml 3.7% sodium citrate/10 ml blood, by Procedure B, 1 ml 1% NA2 EDTA in .7% NaC1/10 ml blood. The tubes were mixed by very gentle inversion and then kept on ice. Analyses were performed within two hours after the last sample was drawn. To obtain platelet-enriched plas-
Uptake and Efflux of Serotonin
87
ma for uptake and efflux studies the appropriate tubes were centrifuged at 82g for 15 minutes. The supernatant solution was drawn off with a widetipped plastic pipet and transferred to polypropylene test tubes for use in Procedure A or B. ProcedureA (seeFigure 1). Ten microliters of serotonin solution containing. 1/~Ci serotonin-3-1'C (specific activity 5.14 gCi/gmol) in water was added to 1 ml citrated platelet-rich plasma to give a final serotonin concentration of approximately 2 X 10 -5 M. The contents were mixed by gentle inversion, 10/~1 was removed and dissolved in .5 ml Soluene (Packard Instrument Inc., Downers Grove, Illinois). Ten milliliters of a P O P O P - - t o l u e n e scintillation mixture was added and radioactivity determined in a Packard 3375 scintillation counter. The remaining solution was
BLOOD DRAWN IN PLASTIC SYRINGE
I platelet counrs 5HT DSH ~er
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Procedures
10,Il ml Antlcoogulent/mlblood A 3.8%Na Citrate or B. 1%EDTA~n0.7%NaCI
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Plotelet counts
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Fig. 1. Outline of procedure.
1 ~
88
Yuwiler, Ritvo, Geller, Glousman, Schneiderman, and Matsuno
incubated at 37~ in a water bath under ordinary atmosphere for 90 minutes, except where otherwise stated. At the end of the incubation period, 95 - m l aliquots of the mixture were placed in a dialyzing support as described below, and two .i-ml aliquots were placed in duplicate polypopylene Beckman Microfuge tubes9 Platelets and plasma were separated by centrifugation at approximately 9,000g for two minutes in a Beckman Model 152 Microfuge. Fifty microliters of supernatant plasma were removed, dissolved in Soluene as .described above, and the count rate determined. The remainder of the supernatant was gently removed, the centrifuge tube cut just above the buffy platelet layer, extraneous moisture, when present, was blotted off by inverting the top onto filter paper, and the entire tip containing the platelet pellet was then dropped into .5 ml Soluene. After dissolution of the peUet overnight, the radioactivity was determined as indicated above. Serotonin uptake was calculated both directly from the counts in the pelletted platelets and indirectly from the remaining counts in the supernatant solution. Total counts in the pellet plus supernatant solution were compared to initial counts in the suspension to monitor losses. Dialyzing supports were constructed of 2.5-cm-diameter polyethylene tubing fitted atone end with a stretched single layer of dialyzing tubing held in place with rubber bands. These supports were most conveniently prepared by cutting the closed ends from the plastic outer containers of 20-ml Monojet disposable syringes and carefully smoothing the cut end to prevent injury to the dialyzing tubing. The dialyzing support containing .5 ml incubated platelet-rich plasma (1.2 mm deep) was immersed to a depth of 2.5 mm in a 2-1iter bath of freshly prepared Krebs-Ringer phosphate buffer (Umbreit, Burris, & Stauffer, 1949) containing 7.6 g sodium citrate and omitting calcium. The bath was kept in continuous circulation with magnetic stirrers. After 90 minutes, duplicate. 1-ml aliquots of the platelet-rich plasma within the supports were removed, centrifuged as described above, and count rates determined. Percent efflux was calculated as 100(l-p,/po) where po represents number of micromoles of radioactive serotonin in the platelets before dialysis and p, the number after dialysis. Procedure B (see Figure 1). Ten microliters of serotonin solution containing. 1 laCi serotonin-3-14C (specific activity, 5.14 laCi//amol) in water was added to 1 ml platelet-rich plasma from EDTA-saline-treated blood and the contents were mixed by gentle inversion. A 10-/al aliquot was removed and its radioactivity determined as described above. The remaining solution was incubated at 37~ in a Dubnoff metabolic shaker at 30 excursions/min under 95% O5/5% CO2 at a flow rate of 8 ft3/min. At the end of 90 minutes, except where otherwise stated, the tubes were removed and immediately cooled in ice. Duplicate .1-ml aliquots were taken,
Uptake and Efflux of Serotonin
89
platelets and plasma separated by centrifugation, and radioactivity determined as described above. Uptake was calculated as described in Procedure A. The remaining .8 ml was centrifuged at 6,880g for 5 minutes, the supernatant plasma aspirated, and the walls of the tube wiped free of remaining supernatant solution. Four drops of 1~/0 EDTA/0.7% sodium chloride solution were added and the tube gently agitated to resuspend the platelet pellet by briefly and iightly flicking the base of the tube. The platelets were then resuspended in .8 ml homologous platelet-free plasma (prepared by centrifuging platelet-rich plasma at 20,000g in a Sorvall RC2B refrigerated centrifuge for 10 minutes), inverted, and then briefly mixed with a vortex mixer. Duplicate .1-ml aliquots again were taken for determination of radioactivity in the supernatant plasma and platelets. The remaining solution was replaced in the incubator under O2/CO~ for an additional 90 minutes after which a final duplicate set o f . 1-ml aliquots were removed, centrifuged, and the radioactivity of the supernatant solutions and the platelets determined. Efflux was calculated as 100(l-p,/po) where p, is the radioactivity in the platelets at the end of the efflux period and po is that at the beginning of the efflux period. A second calculation was made from the radioactivity in the supernatant in which efflux was lO0(1-[(S,-So)/po]) where S0 represents the radioactivity in the supernatant at the beginning of the efflux and S, is that at the end of the efflux period. These procedures are summarized in Figure 1.
RESULTS
Uptake Studies Uptake Studies: Procedure A. Figure 2A illustrates typical serotonin uptake by platelet-enriched plasma at 37~ in an air atmosphere. Uptake reached a s~able plateau in 30-60 minutes, remaining relatively unchanged for the next hour, after which platelet radioactivity slowly fell while supernatant radio– rose. Approximately 30~ of the total radioactivity was platelet bound in 90 minutes with a serotonin bath concentration of 2 X 10-5 M. Uptake approximated 1.27/amol/10 ' ' platelets, in agreement with values obtained by others (Lucas et al., 1971; Boullin et al., 1970). This does not represent the maximum uptake capacity of the platelet, however, for an additional 20% is taken into the platelets if the serotonin concentration of the incubation media was inereased twofold (Figure 2A). Conceivably the additional uptake could result from active displacement of endogenous, unlabeled serotonin by radioactive serotonin rather than accumulation, but this seems unlikely since the uptake curve continued to rise smoothly beyond the maximal endogenous content. Figure 3 presents a scatter dia-
90
Yuwiler, Ritvo, Geller, Glousman, Schneiderman, and Matsuno UPTAKE
B
EFFLUX
14
12
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t
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30
60
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90 120
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180
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Fig. 2. Platelet serotonin uptake and efflux (Procedure A, see text). The curves in A represent the uptake of exogenous "C-labeled serotonin into platelets (open figures and lower curves) and the concomitant loss of serotonin from the plasma (filled circles and upper curve). Uptake curves for the two concentrations of serotonin are shown. The curve for serotonin loss from the plasma is that obtained using a serotonin concentration of 2 X 10-2 M. The curves in B represent the loss of serotonin from platelets (open circles) and supernatant solution (filled circles) in the dialysis tube during the efflux procedure.
gram of the relationship between initial serotonin concentration and ultimate serotonin uptake. The correlation coefficient between these variables is .02, suggesting that uptake is not simply an inverse function of initial serotonin concentration under these conditions. Table I summarizes data obtained from the autistic, normal, and hospitalized nonautistic children using Procedure A. Within the small
Uptake and Efflux of Serotonin
91
populations examined, blood serotonin concentrations were higher in autistic children than in either normals or hospitalized nonautistic subjects. The differences reached statistical significance only between the autistic and normal groups. However, these could be because the samples were so small, because hospitalization may effect blood serotonin concentration, because of the diagnostic nonspecificity of elevated serotonin concentrations, or because of the diagnostic heterogeneity of the populations. Blood platelets were also higher in the autistic group but differences were smaller than previously observed and were not statistically significant. As might be expected from these results, serotonin/10" platelets were also significantly higher in aut9 than in comparison populations but the difference between autistics and hospitalized controls failed to reach statistical significance. I
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Fig. 3, Relationship between basal platelet serotonin content and serotonin uptake under condition of procedure A. Open circles ( o ) are values for normal controls. Open triangles ( A ) are values for hospitalized nonautistic controls, and filled circles (*) are values for autistic subjects. The equation for the least-square line is y = 0 . 0 4 7 x + 1 . 4 0 8 , and the correlation coefficient is .02.
92
Yuwiler, Ritvo, Geiler, Glousman, Schneiderman, and Matsuno Table I, Basal Serotonin, Serotonin Uptake, and Efflux by Platelets
A
Group Normal Autistic Hospital controls
Basal (mean • SE, and N)
Age in months (mean -+SE)
Serotonin (#g/ml)
Platelets (10S/mm s)
5HT/platelet (~mol/1011)
95.5 -+ 25 60.2 • 13 69.4 • 20
.205 -+ .017 (12) .273 • .030(12) a .240 -+ .014(14)
310 -+ 12(14) 333 -+ 23(12) 324 -+21(12)
.369 +- .027(12) .516 +- .060(12) a .436 • .033(12)
B
Group Normal
Procedure A (mean _+SE, and N) Uptake (#mol 5HT/1011 Efflux platelets) (% change)
Procedttre B (mean _+SE, and N) Uptake (btmol 5HT/1011 Efflux platelets) (% change)
1.452 +_.070(11) -22.8 -+ 3.3(11) b 1.482 • .016(11) 2.01 • 2.8(12) b .49 _+ .8(12) c
Autistic
1.221 • .092(11) -22.3 +- 3.2(11) b 1.400 +- .050(11) -.56 • 3,5(12) b .52 • 1.1(12) c Hospitalcontrols 1.309 -+ .020(13) -23.7 -+3.6(13) b Values are given as mean and standard error. The number of patients within each g~oup is indicated within parentheses. Significantly different from normals; p < .05. Calculated from difference in platelet radioactivity between beginning and end of efflux period. CCalculated from dffferences in supernatant radioactivity betwš beginning and end of efflux period.
~
I n a g r e e m e n t with t h e r e p o r t o f Siva S a n k a r (1963), u p t a k e b y p l a t e lets f r o m autistic c h i l d r e n in this h a l f o f t h e s t u d y t e n d e d to be l o w e r t h a n t h o s e f r o m t h e o t h e r g r o u p s . H o w e v e r , t h e d i f f e r e n c e was n o t s t a t i s t i c a l l y significant a n d was n o t o b s e r v e d w h e n r e e x a m i n e d b y P r o c e d u r e B (see below). T h e d i f f e r e n c e was also d i m i n i s h e d w h e n u p t a k e was c a l c u l a t e d f r o m t h e loss o f r a d i o a c t i v i t y o f t h e s u p e r n a t a n t d u r i n g i n c u b a t i o n r a t h e r t h a n f r o m t h e g a i n in p l a t e l e t r a d i o a c t i v i t y . B a s œ o n c h a n g e s in t h e s u p e r n a t a n t , p l a t e l e t u p t a k e was 1 . 2 6 5 - . 1 7 7 / a m o l / 1 0 " platelets ( m e a n -+ SE) for autistics, 1.346 +-- .155 for h o s p i t a l i z e d c o n t r o l s , a n d 1.322 +- .160 f o r n o r m a l s . Because o f the ease o f s a m p l i n g , s u p e r n a t a n t c o u n t s are o f t e n u s e d in u p t a k e studies. This has the d i s a d v a n t a g e t h a t c a l c u l a t i o n s a r e b a s e d u p o n the d i f f e r e n c e b e t w e e n t w o large n u m b e r s a n d is s u b j e c t t o t h e e r r o r s in b o t h . D i r e c t e s t i m a t i o n s o f r a d i o a c t i v i t y in platelets a v o i d this s o u r c e o f e r r o r b u t surfer f r o m p o s s i b l e s a m p l i n g e r r o r s d u e t o t h e p e n c h a n t o f p l a t e lets to a d h e r e to surfaces a n d f r o m v a r i a b l e e n t r a p m e n t o f s u p e r n a t a n t in the p l a t e l e t pellet.
Uptake and Efflux of Serotonin
93
Uptake Studies: Procedure B. A typical uptake curve under the conditions described in Procedure B is presented in Figure 5A while Figure 5B presents the temporal changes in platelet content following resuspension o f loaded platelets in h o m o l o g o u s platelet-free plasma. Serotonin uptake continued to rise over the 90-minute incubation period and had not reached plateau values by the end of that time. Nonetheless, 90 minutes was selected as the end point o f uptake time to c o n f o r m as closely as possible to the procedure described by Boullin et al. (1970). The results obtained are summarized in Table IB. Serotonin uptake by platelets in Procedure B was slightly higher than that observed using Procedure A. The groups did not differ on this measure and uptakes were similar to those reported by Boullin et al. (1970). As seen in Figure 6, there was I
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Fig. 4. Relationship between serotonin uptake by platelets and serotonin loss from platelets during dialysis (Procedure A). Open circles represent values for normal control children, open triangles are those for nonautistic hospitalized controls, and fiUed circles are those for autistic subjects. The equation for the least-squares line is y = 12.5 x + 5.3, and the correlation coefficient is .39.
94
Yuwiler, Ritvo, Geller, Glousman, Schneiderman, and Matsuno 20 UPTAKE
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Fig. 5. Platelet serotonin uptake and efflux. A represents the uptake of exogenous ;'C-labeled serotonin into platelets (open circles) and the concommitant loss from platelets (filled circles)under the incubation conditions described in Procedure B. B represents the leakage from platelets (open circles) and the increase in plasma upon resuspension and incubation of serotonin-loaded platelets with homologous platelet-free pJasma (filled circles) as described in Procedure B. a g a i n little r e l a t i o n s h i p b e t w e e n s e r o t o n i n u p t a k e a n d b a s a l platelet serotonin content.
Efflux Studies Efflux Studies: Procedure A. A typical curve for the efflux o f seroton i n f r o m p r e l o a d e d platelets as m e a s u r e d using the dialysis t e c h n i q u e is s h o w n i n Figure 2B. F i n a l platelet s e r o t o n i n c o n c e n t r a t i o n is 60--100070 o f
Uptake and Efflux of Serotonin
95
the initial loaded level. Presumably, the initial loss is diffusible serotonin from the cytoplasm while the stabilized level consists of vesicular-bound serotonin.. Again, however, loss is n o t a simple function of initial serotonin content. Thus the scatter diagram relating uptake to efflux (Figure 4) indicates that only 16070 of the variance, at most, can be accounted for by the platelet serotonin content at the end of the uptake period. As seen in Table 1B, groups did hOt differ in serotonin uptake using this procedure. Efflux Studies: Procedure B. Efflux of serotonin from platelets in Procedure B was minimal whether calculated from the loss of platelet serotonin before and after reincubation in homologous plasma or from the increase in supernatant radioactivity. However, a mean loss of radioactivity of 26.907o for platelets of autistic children and 27.7070 for platelets of normals was observed during the concentration and resuspension of preloaded platelets. This loss in platelet radioactivity is equivalent to that seen in ProI
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BASAL SEROTONIN CONTENT (#mol serotonin/lO 11 platelets) Fig. 6. Relationship between basal serotonin platelet content and serotonin uptake under the incubation conditions of Procedure B. Open circles represent values for normal control children; filled circles, values for autistic children. The equation of the least-square line is y = 1.534 x + .912, and the correlation coefficient is .34.
96
Yuwiler, Ritvo, Geller, Glousman, Schneiderman, and Matsuno
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Fig. 7. Relationship betwee¡ serotonin uptake and serotonin efflux from platelets under the condition described for Procedure B. Open circles represent values for normal controls, while filled circles are those for autistic subjects. The equation for the least-squares fit of the data is y = -1.83 x + 3.38, and the correlation coefficient is -.27.
cedure A during dialysis. As seen in Table IB, efflux was the same for both groups by this procedure. Again, the scatter diagram relating efflux and uptake, Figure 7, suggests that these two processes are only minimally related.
DISCUSSION
Several variables could contribute to differences others have observed in serotonin uptake and efflux from platelets of autistic and normal children. Among these are diagnostic criteria, age, prior drug treatment, and experimental methodology. The diagnostic comparability of the experimental groups is particularly difficult to assess. Siva Sankar et al. (1963) labeled their experimental population autistic/childhood schizophrenic but did not specify their diagnostic criteria. Lucas et al. (1971) used the criteria
Uptake and Efflux of Serotonin
97
of Bender (1968) and Rabinovich, Lucas, Ingram, and Shaw (1965) to define their experimental population as schizophrenics while Boullin et al. (1970, 1971) used the experimental history checklist of Rimland (1968) to identify patients as havingprimary infantile autism. The criteria used in the present study have been detailed in the liter” (Ornitz & Ritvo, 1968) and are summarized in the Methods section. The prior drug status of the patients also varies between studies. Experimental and comparison subjects in Siva Sankar's study (1963) were drug free for 7-10 days prior to the experiment. Those in Lucas' study (1971) were drug free for "at least three days.', Boullin's (1970) patients were drug free for ai least one month. In the present study, none of the subjects had ever received major tranquilizers and ail of them were free of ail medication for at least three weeks prior to testing. Age is also an important experimental variable since both blood serotonin content and platelet counts decrease with age until adult levels are reached at age 14 (Ritvo et al., 1970). Siva Sankar's patients (1963) were 7-15 years of age and differences in uptake were observed only in the 10-15-year-old group. Lucas' patients (1971) ranged from 8 to 15 years of age, with a mean of 12.9. Boullin's patients (1970) ranged in age from 4 to 14 years. In the present study the autistics' mean age was 60.2 months (range 3-6 years), the normals' was 97.5 months (range 5-12 years), and the hospitalized controls 69.4 months (range 5.5-11.5 years). The effects of anticoagulents and temperature on platelet function and morphology are known to be complex. Siva Sankar et al. (1963) used heparin as anticoagulent, Lucas et al. (1971) used sodium citrate a t a final concentration of 1.8o70, and Boullin et al. (1970) used 1~ EDTA. In the present study, citrate at a final concentration of .38~ was used for Procedure A, and 1.8~ EDTA was used in Procedure B. Platelets in citrate are generally considered to maintain normal morphology. Although citrate inhibits dumping, clumping can occur with low levels of citrate (or heparin). Further, platelet aggregation in citrate solutions is induced by sudden chilling, particularly if followed by stirring during rewarming (Kattlove & Alexander, 1971). Indeed, resuspension of packed, chilled, citrated platelets leads to massive loss of label. Chilling itself leads to platelet sphering and an increase in platelet volume, while r and washing often result in platelet damage and release of ADP. Platelets treated with EDTA do hOt clump upon centrifugation and chilling, although they may be otherwise damaged by the procedure. However, EDTA alone causes platelet rounding, swelling, and an increase in platelet volume. In the studies discussed here, Siva Sankar et al. centrifuged and washed heparinized serotoninloaded platelets before lysis and counting. Lucas et al. cooled and centrifuged citrated platelets and maintained them a t . 1~ before rewarming and dialyzing. Boullin used EDTA, chilling, and centrifugation, before resus-
98
Yuwiler, Ritvo, Geller, Glousman, Schneiderman, and Matsuno
pension and reincubation at 37~ These differences in technique might yield platelet preparations with different metabolic transport properties. In summary, platelets from young normals, autistics, or hospitalized controls did not differ in serotonin uptake or efflux in two procedures which differed in anticoagulent, gas phases, temperature, and platelet treatment. As detailed elsewhere (Ornitz & Ritvo, 1968; Ornitz, 1973) confusion exists in the literature over the nature and diagnosis of autism and while we believe that a neuropathophysiologic substrate is involved in this disease, the present results do not support the view that this is reflected in the monamine transport system of platelets.
REFERENCES Bender, L. Childhood schizophrenia: A review. International Journal of Psyehiatry, 1968, 5, 211. Boullin, D. J., Coleman, M., & O'Brien, R. A. Abnormalities in platelet 5-hydroxytryptamine efflux in patients with infantile autism. Nature, 1970, 226, 371-372. Boullin, D. J., Coleman, M., O'Brien, R. A., & Rimland, B. Laboratory predictions of infantile autism based on 5-hydroxytryptaminœ efflux from blood platelets and their correlation with the Rimland E2 score. Journal of Autism and Childhood Schizophrenia, 1971, 1, 63-71. Coleman, M. (Ed.). Serotonin in Down's Syndrome. New York: American Elsevier, 1973. Chap. 9. Kanner, L. Autistic disturbances of affective contact. Nervous Child, 1943, 2, 217-250. Kattlove, H. E., & Alexander, B. The effect of cold on platelets: Cold-induced platelet aggregation. Blood, 1971, 38, 39-48. Lucas, A. R., Warner, K., & Gottlieb, J. S. Biological studies in childhood schizophrenia. Biological Psychiatry, 1971, 3, 123-128. Ornitz, E. M. Childhood autism: A review of the clinical and experimental literature (medical progress). California Medicine, 1973, 118, 21-47. Ornitz, E. M., & Ritvo, E. R. Perceptual inconstancy in early infantile autism. Archives of General Psychiatry, 1968, 18, 76-98. Pletscher, A. Metabolism, transfer and storage of 5-hydroxytryptamine in blood platelets. British Journal of Pharmaeology and Chemotherapy, 1968, 32, 1-16. Rabinovitch, R. D., Lucas, A. R., Ingram, W., & Shaw, C. R. Childhood schizophrenia: Evolution to adulthoocl. Presented at American Orthopsychiatric Association Annual Mš New York, 1965. Rimland, B. On the objective diagnosis of infantile autism. Acta Paedopsyehiatriea, 1968, 35, 1946-1961. Ritvo, E. R., Yuwiler, A., Geller, E., Ornitz, E. M., Saeger, K., & Plotkin, S. Increased blood serotonin and platelets in early infantile autism. Archives ofGeneral Psyehiatry, 1970, 23, 566-572. Siva Sankar, D. V., Cates, N., Broer, H., & Sankar, D. B. Biochemical parameters of childhood schizophrenia (autism) and growth. In J. Wortis (Ed.), Recent Advances in Biologieal Psychiatry (Vol. 5). New York: Plenum, 1963, pp. 76-83. Umbreit, W. W., Burris, R. H., & Stauffer, T. D. Mannometric techniques. Minneapolis: Burgess, 1949. Yuwiler, A., Plotkin, S., Geller, E., & Ritvo, E. Rapid accurate procedure for the determination of serotonin in whole human blood. Biochemical Medicine, 1970, 3, 426-436.
